ORIGINAL ARTICLE

A phylogenetic and morphological study of Polystachya sect.Superpositae (Orchidaceae) with description of a new speciesfrom Cameroon

Joanna Mytnik-Ejsmont • Dariusz L. Szlachetko •

Przemysław Baranow • Marcin Gorniak

Received: 14 January 2013 / Accepted: 27 May 2013

� The Author(s) 2013. This article is published with open access at Springerlink.com

Abstract Polystachya section Superpositae is a group of

montane and submontane African epiphytes with thirteen

species (including the newly described in this paper).

During a scientific expedition to Bamenda Highland,

Cameroon a new species, Polystachya bamendae, was

found and it is described here. The new entity was com-

pared with 398 herbarium specimens representing the

section, including the type collections. The molecular

analyses of the ITS and matK strongly support the results

of morphological studies and reveal close relationship

between the new species and the species of the section

Superpositae. Two hypotheses supporting function of the

matK gene in the examined species are presented. The

phylogenetic and morphological relation of Polystachya

bamendae to P. lindblomii and P. superposita is discussed.

The distribution range of the section is analyzed and a

discussion about the distribution of the species is presented.

A distribution map of the section and dichotomous key to

the species of Superpositae are provided. The occurrence

of the species of the section in two Biodiversity Hotspots

(Guinea Forests of West Africa and Eastern Afromontane)

is discussed.

Keywords Africa � ITS � matK � Morphology �Taxonomy

Introduction

The genus Polystachya Hook. includes 233 species

worldwide, of which nearly 90 % occurs in Africa (Myt-

nik-Ejsmont 2011). Representatives of the genus are sym-

podial, epiphytic, lithophytic or occasionally terrestrial

herbs with terminal inflorescences growing always out of

the top of the pseudobulb, with non-resupinate flowers. The

flowers, predominantly small, are characterized by a

mentum, which is a fusion of lateral sepals and the column

foot, and three-lobed lip with usually oblong callus.

The section Superpositae Kraenzl. is one of 13 sections

of Polystachya (Mytnik-Ejsmont 2011). It was described

by Kraenzlin in the first monographic treatment of the

genus (1926). The representatives of the section are mon-

tane and submontane epiphytes characterized i.e., by

unique vegetative characters—fusiform pseudobulbs united

in an erect or pendulous sympodium, each succeeding one

arising from the middle or apical part of the proceeding

one, 2–8-leaved, short unbranched or paniculate inflores-

cence, free part of gynostemium 1–2.5(5) mm high, and

prominent column foot 4–5 mm long.

The section is divided into three subsections (Mytnik-

Ejsmont 2011). The nominal subsection includes five spe-

cies, P. eurygnatha Summerh., P. fusiformis (Thouars)

Lindl., P. simplex Rendle, P. stewartiana Geerinck, and P.

superposita Rchb.f. They are plants with pseudobulbs

3 mm in diameter at least, narrowly elliptic leaves,

paniculate, 20–80-flowered inflorescence, lip shortly and

broadly clawed, most often without any callus. Within the

subsection Spatellae Mytnik there is classified seven spe-

cies, P. spatella Kraenzl., P. ruwenzoriensis Rendle, P.

lindblomii Schltr., P. pudorina P.J.Cribb, P. tridentata

Summerh., P. aethiopica P.J.Cribb and newly described

P. bamendae Szlachetko, Baranow & Mytnik. They can be

J. Mytnik-Ejsmont (&) � D. L. Szlachetko � P. Baranow

Department of Plant Taxonomy and Nature Conservation,

The University of Gdansk, ul. Wita Stwosza 59, 80-308 Gdansk,

Poland

e-mail: dokjom@univ.gda.pl

M. Gorniak

Department of Molecular Evolution, The University of Gdansk,

Wita Stwosza 59, 80-308 Gdansk, Poland

123

Plant Syst Evol

DOI 10.1007/s00606-013-0856-0

defined by possession of slender stems up to 3 mm in

diameter, scarcely thickened, narrowly oblong ligulate to

linear leaves, unbranched inflorescence 1–16-flowered

(most often consisting of 3–10 flowers), and long-clawed

lip furnished with a fleshy callus. The subsection Kerme-

sinae Mytnik is a monotypic taxon represented by P. ker-

mesina Kraenzl., one of the showiest species within the

subtribe. The species is atypical for Superpositae for its

flower color and lip structure, however, due to its super-

posed, slender, 2–3-leaved pseudobulbs, linear leaves,

short 3–5-flowered inflorescence, gynostemium structure

and distribution range as well, Mytnik-Ejsmont (2011)

decided to keep P. kermesina in Superpositae.

The section was revised once by Cribb (1979). The author

presented a synopsis of the group and proposed three infor-

mal subgroups within the section. He included six species to

the first group (P. spatella, P. ruwenzoriensis, P. lindblomii,

P. pudorina, P. tridentata, and P. aethiopica), they were

characterized by slender cylindrical stems scarcely pseudo-

bulbous, narrow, grass-like leaves, medium-sized flowers,

unbranched inflorescence, and the long-clawed lip furnished

with a fleshy simple or lobed porrect callus. The plants with

stouter pseudobulbs, broader, narrowly elliptic leaves, a

paniculate inflorescence, and small flowers with a shortly

clawed lip with or without a small unlobed callus were

included in the second group (P. simplex, P. eurygnatha, P.

fusiformis, and P. superposita). The last monotypic group

was represented by Malgasian P. oreocharis Kraenzl.

The latter species, however, was excluded from the

section by Mytnik-Ejsmont (2011). It was classified within

Superpositae by many authors (Kraenzlin 1926; Cribb

1979, 1984) though it is a completely odd taxon. This

Malgasian endemic is characterized by non-pseudobulbous

stems completely covered by the imbricating leaf bases,

many distichously arranged leaves, geniculate lip with

strongly undulate margins and horn-shaped basal callus.

Apart from Polystachya superposita and P. bamendae,

the novelty described in this paper, being endemic to

Cameroon Highlands and P. fusiformis being widely dis-

tributed throughout tropical and subtropical Africa and

Madagascar, vast majority of the species of Superpositae

are confined to East African Rift System (Eastern Afro-

montane Biodiversity Hotspot), which is a center of high

concentration of the species of the section. They occur

mainly at higher elevations, most often from 850 to

3,200 m above sea level from Ethiopia to Mozambique.

Five East African species of the section (P. eurugnatha, P.

kermesina, P. ruwenzoriensis, P. stewartiana, and P.

tridentata) are confined in their distribution to the Alber-

tine Rift (=Western Rift Valley), the western branch of the

East African Rift. The Albertine Rift, the region of a large

number of endemic species, has been identified as an

ecoregion by the World Wildlife Fund and a Biodiversity

Hotspot by Conservation International (2005). Another

representative of the section, Polystachya pudorina, is an

endemic to part of the eastern branch of the East African

Rift, the Usambara Mountains, which are unique ancient

mountain region covered with the tropical forest. Due to a

lack of glaciations and a relatively consistent climate, the

rainforest of Usambaras has gone through a long-term

evolution resulting in an impressive amount of endemism

(Lovett 1993). Polystachya simplex and P. lindblomii,

occur in both branches of the East African Rift along its

whole length, from Ethiopian Highlands to Mozambique.

Polystachya spatella, though known in both Western and

Eastern Rift Valley, is confined in its distribution to the

region of Victoria Lake. The species is one of the very few

polystachyas growing above 3,000 m.a.s.l. It was even

found above the evergreen forest belt, in the bamboo zone

in Ruwenzori Mountains. The only species occurring

exclusively in Ethiopia is Polystcahya aethiopica, being

found in two ecoregions (WWF 2012): the Ethiopian

montane forests (up to 1,800 m elevation) and the Ethio-

pian montane grasslands and woodlands (above 1,800 m).

Today the section includes thirteen species (including

the new entity). The new species described here was found

during a scientific expedition to Cameroon, in which

Szlachetko and Baranow have been conducted taxonomic

research of Orchidaceae. The country is among the top ten

countries in Africa for biodiversity due to its montane

forests, where a high number of endemic plant species

occur (BirdLife International 2010) and to its strong lati-

tudinal gradient of precipitation. Now the section Super-

positae is represented by three species in Cameroon.

Material examined

Morphological study. The study presented here is based on

the examination of the plant material collected in the field

and 398 herbarium specimens from fifteen herbaria: B,

BM, BR, BRLU, C, HBG, K, L, MA, MO, P, UGDA, W,

WAG and YA (acronyms according to Thiers 2008, con-

tinuously updated). We observed some living plants in

WAG and C. The specimen representing the new species

was found in south-east of the Bamenda Highlands, about

2 h walking from the village Big Babanki to Czech Field

Station.

DNA material. DNA sequences were downloaded from

GenBank (http://www.ncbi.nlm.nih.gov/genbank/). Two

data matrices, ITS and rps16-matK were taken from Pop-

Set database no 298263940 (Russell et al. 2010a) and

270484601 (Russell et al. 2010b), respectively. The rps16-

matK data matrix was trimmed for part of matK gene only

and then analyzed. Four species (Adrorhizon purpurascens,

Sirhookera lanceolata, Bromheadia finlaysoniana, and B.

J. Mytnik-Ejsmont et al.

123

srilankensis) were selected as outgroup for matK and two

species (Adrorhizon purpurascens and Sirhookera lanceo-

lata) were selected as outgroup for ITS. In addition, three

species (ITS) were taken from GenBank: Polystachya

spatella HM018555, P. campyloglossa HM041027, P.

lawrenceana HM018549. One species (ITS), P. fusiformis

(id 068) was taken from data matrix from Anton Russell.

Plant material for new species was collected during field

expedition in 2011 to Cameroon. DNA from this specimen

was extracted from silica dried leaves (Chase and Hillis

1991) using DNA easy Plant Mini Kit (A&A Biotechnol-

ogy) following manufacturer protocol. ITS and part of

matK gene (approximately 1,300 bp) were sequenced.

Sequences were deposited in GenBank under accession

number KC339535 (matK) and KC339536 (ITS).

Methods

DNA amplification and sequencing. Polymerase chain

reaction (PCR) amplifications were carried out in a total

volume of 25 ll containing 2.5 ll 109 buffer, 1 ll 25 mM

MgCl2, 1 ll 5 mM dNTPs, 0.5 ll of 10 lM of each

primers and 1 U of Red Perpetual DNA polymerase (Eurx,

Poland) and H2O. The ITS region (ITS1-5.8S-ITS2) was

amplified via PCR using the primers 101 AB and 102 AB

(Douzery et al. 1999), the matK was amplified with the

following two primers: 19F (Molvray et al. 2000) and

1,326R (Cuenoud et al. 2002). The PCR cycling conditions

for ITS and matK were template premelting at 94 �C for

3 min followed by 30 cycles of denaturation at 95 �C for

45 s, primer annealing at 52 �C for 1 min, followed by

primer extension at 72 �C for 90 s. Final extension step

was of 7 min at 72 �C. Amplified products were cleaned

with High Pure PCR Product Purification Kit (Roche

Diagnostic GmbH, Mannheim, Germany) following man-

ufacturer protocol. Cycle sequencing was carried out using

Big Dye Terminator v 3.1 Cycle Sequencing Kit (Applied

Biosystems, Inc., ABI, Warrington, Cheshire, UK) with the

same primers used for PCR amplification: 2.0 ll of 59

sequencing buffer, 1 ll of Big Dye terminator with 1.5 ll

of 10 lM primer (1.5 pmol), 1–2 ll of amplified product

(30–90 ng/ll), and H2O in a total of 10 ll reaction volume.

Cycle sequencing conditions were as follows: 25 cycles

each with 15 s denaturation (94 �C), 5 s annealing (50 �C),

and 4 min elongation (60 �C). The sequences were gener-

ated on an ABI 3720 automated capillary DNA sequencer.

Both strands were sequenced to assure accuracy in base

calling. Sequence Navigator (ABI) was used to edit the

sequences, and the two complementary strands were

assembled using AutoAssembler (ABI).

Sequence alignment and phylogenetic analyses. The ITS

and matK sequences were aligned by eye using Seaview

(Galtier et al. 1996). Maximum parsimony analyses were

undertaken on two matrices separately and combined

analyses using heuristic searches in PAUP* version beta 10

(Swofford 2000) with tree-bisection-reconnection (TBR)

branch swapping and the MULTREES (holding multiple

trees) option in effect with 1,000 replicates of random

sequence addition, but saving only 10 trees for each rep-

licate to reduce the time spent in swapping large numbers

of suboptimal trees. Standard tree parameters were esti-

mated (tree length, consistency index, CI, and retention

index, RI). Internal support of clades was evaluated using

non-parametric bootstrapping (Felsenstein 1985) with

1,000 replicates and the same settings as above, except for

simple sequence addition. All characters were treated as

unordered and equally weighted (Fitch 1971).

Morphological study. Standard procedure of preparing

the herbarium material to facilitate stereomicroscopic

observation was applied. The following vegetative charac-

ters of individual plants were analyzed: stem (height, shape,

presence of glandular hairs), leaves (number, size, shape),

sheaths (number, shape, size), inflorescence (size, density),

floral bracts (size, shape, presence of glandular hairs) flow-

ers, taken from the middle part of the inflorescence (presence

of glandular hairs, size of pedicel and ovary, height and shape

of mentum (spur), size and shape of lateral sepals, dorsal

sepal, petals, and lip), as well as gynostemium. Particular

parts of the flower were boiled, dissected, measured and

drawn under a stereomicroscope. The results were then

analyzed and compared with the type material, diagnoses

and original illustrations. The database of the drawings and

photographs of all studied specimens is available in the first

author’s archives. For all species type material was available.

Results

Statistics for ITS and matK and combined data matrices are

separated by ‘‘/’’. The number of analyzed taxa was 75/129/

15, respectively. The aligned matrix comprised 860/1,326/

2,158 characters of which 295/263/295 were variable and

195/162/120 were potentially parsimonial informative. The

number of the most parsimonious trees were [10,000 for

ITS and matK and 3 for combined analysis. Tree length

was 702/414/360, consistency index (CI) = 0.6/0.7/0.7,

and retention index (RI) = 0.8/0.9/0.8. One of the most

parsimonious trees is depicted in Figs. 1, 2 and 3. Boot-

strap support (BS) C50 % is given for supported clades

below branches. Clades that collapse in the strict consensus

tree are marked by an arrow. Both analyses indicate close

relationships between new species and Polystachya section

Superpositae. The bootstrap support for mentioned clade is

low (BS \50/=50/56). Except P. lindblomii, all sequences

from the Superpositae clade in matK gene possess 19 bp

A phylogenetic and morphological study

123

indel (duplication event). The indel occurrence causes a

change of frameshift and as a natural consequence, an

activation of the stop codon. The movement of the

frameshift by one nucleotide downstream in the species

possessing the indel enables an obtainment in silico the

amino-acid sequence, which is homological to the

sequences of the rest of the species and it does not have the

stop codon within the gene sequence.

Taxonomic treatment

Polystachya Hook. sect. Superpositae Kraenzl. subsect.

Spatellae Mytnik

Polystachya bamendae Szlachetko, Baranow & Mytnik,

sp. nov.

Type: Cameroon. North West Province: Bamenda

Highlands about 2 h walking from the village Big Babanki

to the Czech Field Station, in degraded secondary forest, ca

2–3 m above the ground on Eucalyptus trunk, elev.

2,150 m, 11–26 Nov 2011, fl. Sep–Oct 2012, Szlachetko &

Baranow 9465 (holotype: UGDA; drawing of the holotype:

BRLU, K, YA).

Polystachya bamendae is related to P. lindbomii, but the

former species has the fractiflex rachis, prominent lip lat-

eral lobes and the lip middle lobe 3 times wider than long.

Etymology: In reference to the place where the type

specimen was collected.

En epiphytic plant up to 20 cm long. Pseudobulbs

7–11 cm long, 0.3–0.6 cm in diameter, fusiform, each

succeeding one arising from the middle or apical part of the

proceeding one. Leaves up to 6.5 cm long, 0.7 cm wide,

narrowly lanceolate to linear-lanceolate, shortly acuminate.

Inflorescence up to 8 cm long, branched, 4–20-flowered;

rachis fractiflex, pubescent. Floral bracts 3 mm long, 4 mm

wide, caudate. Flowers sparsely pubescent, fleshy, green

with white apices of petals, lip yellow-white with yellow

callus in the center. Pedicel and ovary 3–4 mm long,

sparsely pubescent. Dorsal sepal 4 mm long, 2.8 mm wide,

oblong-elliptic, caudate, apex 1.5 mm long. Lateral sepals

4.5–5 mm long, 2 mm wide, saccate, lance-ovate, caudate,

apex 1–1.5 mm long. Mentum (spur) deeply bilobed, up to

4 mm long, 2 mm wide, rounded, saccate. Petals 3.5 mm

long, 1 mm wide, narrowly elliptic, acute. Lip 4.2–6 mm

long, 4–6 mm wide, 3-lobed, glabrous, strongly fleshy in

the middle, squeezed in the middle on the outer surface,

Fig. 1 One of the most parsimonious trees in ITS analysis (L = 702,

CI = 0.6 and RI = 0.8) for Polystachyinae. New species is high-

lighted in bold. The numbers above branches are Fitch branch lengths.

Bootstrap percentages [50 are given for supported clades below

branches. Arrows indicate clades that collapse in the strict consensus

tree

J. Mytnik-Ejsmont et al.

123

unguiculate, claw 2.5 mm long, 1.8 mm wide, ribbon-like,

strongly fleshy along the midnerve; lateral lobes 2–2.5 mm

long, 1.3–20 mm wide, widely obovate, obtuse, widely

divergent; the middle lobe 2 mm long, 4.5–6 mm wide,

transversely widely oblong, crenulate on margins, 4-lobed,

emarginate with an apex 0.5–0.6 mm long on outer

surface; the callus 1–2 mm long, 1 m high, fleshy bilobed

ridge along the midnerve in the middle of the lip. Column

foot 2–2.5 mm long, column 1.4–2 mm long, stout, anther

1.1 mm long and wide, beak-like, anther partitions prom-

inent, lemon-shaped, 0.5 mm long, 0.3 mm wide; rostel-

lum remnant retuse (Fig. 4).

Fig. 2 One of the most parsimonious trees in matK analysis

(L = 414, CI = 0.7 and RI = 0.9) for Polystachyinae. The new

species is highlighted in bold. The numbers above branches are Fitch

branch lengths. Bootstrap percentages [50 are given for supported

clades below branches. Arrows indicate clades that collapse in the

strict consensus tree

Fig. 3 One of the most

parsimonious trees in combined

(matK ? ITS) analysis

(L = 360, CI = 0.8 and

RI = 0.9) for Polystachyinae.

The new species is highlighted

in bold. The numbers above

branches are Fitch branch

lengths. Bootstrap percentages

[50 are given for supported

clades below branches—

combined/ITS/matK,

respectively. Arrows indicate

clades that collapse in the strict

consensus tree

A phylogenetic and morphological study

123

The morphological differences between the new species

and the other taxa classified within the subsection Spatellae

are presented in the Table 1.

Key to the species of Polystachya section Superpositae:

1. Flowers orange to scarlet, fleshy, almost flat when

opened, lip geniculate Polystachya kermesina

1.* Flowers white, yellow, green, pink or purple, not

widely opening, lip not geniculate 2

2. Inflorescence paniculate, lip shortly clawed 3

2.* Inflorescence simple, lip long-clawed 7

3. Flowers fairly large, mentum 4 mm long or more,

lip 15–18 mm long Polystachya stewartiana

3.* Flowers small, mentum less than 4 mm long, lip

2–6 mm long 4

4. Bracts 1–2.5 mm long, mentum less than 2 mm long,

rounded 5

4.* Bracts 3–7 mm long, mentum more than 2.5 mm

long, conical 6

5. Pseudobulbs 2–4-leaved, leaves linear-lanceolate,

4–9 cm long, lip with a callus Polystachya suerposita

5.* Pseudobulbs 4–7-leaved, leaves oblong-lanceolate,

5–16 cm long, lip without a callus Polystachya

fusiformis

6. Sepals long acuminate, lip lateral lobes oblong,

middle lobe acuminate Polystachya simplex

Fig. 4 Polystachya bamendae Szlachetko, Baranow & Mytnik

a habit, b inflorescences and leaves, c leaf, d floral bract, e flower,

f mentum, g lateral sepal, h dorsal sepal, i petal, j flower lacking

lateral sepals, k lip, l trichomes on sepals, m rostellum remnant,

n anther top view, o anther bottom view, p anther partition,

r gynostemium

J. Mytnik-Ejsmont et al.

123

6.* Sepals acute or subacute, lip lateral lobes obscure,

middle lobe subacute or shortly mucronate Polystac-

hya eurygnatha

7. Ovary densely pubescent 8

7.* Ovary glabrous or very sparsely pubescent 11

8. Leaf more than 6.5 mm wide 9

8.* Leaf less than 6 mm wide 10

9. Lip callus 3-toothed, lip surface glabrous Polystachya

tridentata

9.* Lip callus simple, lip pubescent Polystachya

aethiopica

10. Flowers yellow-green, petals linear Polystachya

lindblomii

10.* Flowers pink or white with a crimson column, petals

ovate-elliptic Polystachya pudorina

11. Lip middle lobe markedly longer than wide Polystac-

hya ruwenzoriensis

11.* Lip middle lobe markedly wider than long 12

12. Mentum more than 4.5 mm long, slightly bifid, lip

middle lobe oblate Polystachya spatella

12.* Mentum up to 4 mm long, deeply bilobed, lip middle

lobe transversely widely oblong Polystachya

bamendae

Polystachya bamendae, as well as P. superposita, are

species endemic to submontane and montane forest of

Cameroon (a part of Guinea Forests of West Africa Bio-

diversity Hotspot), (Figs. 5, 6). The representative of the

newly described species was collected in the south-eastern

part of the Bamenda Highlands, where the remaining pat-

ches of montane forest are very small (up to 500 ha,

BirdLife International 2010), but biologically very impor-

tant. As the narrowly endemic species, both Polystachya

bamendae and P. superposita, should become a subject of

very careful conservation action. Cameroonian Highland

forests, where the species occur, are being cleared for

firewood, timber and to create farmland. The Kilum-Ijim

Forest, where two specimens of P. superposita were found,

is the largest remaining patch of Afro-montane forest in

West Africa being a very important area for biodiversity.

Despite the same distribution range, both species are rather

easy to distinguish morphologically. Polystachya bamen-

dae is characterized by the fractiflex rachis, pubescent

flowers, prominent deeply bilobed mentum up to 4 mm

long and a fleshy clawed lip. The lip lateral lobes of the

latter species are outspread, up to 2.5 mm long and 2 mm

wide, the lip middle lobe is at least twice wider than long.

Table 1 The differences between the species of Polystachya Hook. section Superpositae Kraenzl. subsection Spatellae Mytnik

Polystachya

aethiopica

Polystachya

lindblomii

Polystachya

pudorina

Polystachya

ruwenzoriensis

Polystachya tridentata Polystachya

spatella

Polystachya

bamendae

Leaf

width

(mm)

6–8.5 (1)2–4 1.5–3 4–11 4–11 2.5–6 Up to 7

Pedicel

and

ovary

Densely

pubescent

Densely

pubescent

Densely

pubescent

Glabrous or

sparsely

pubescent

Densely pubescent Glabrous Sparsely

pubescent

Petals Linear-

lanceolate,

acute

Linear to oblong-

elliptic, acute

Elliptic,

shortly

clawed,

obtuse to

rounded

Spathulate,

acute

Oblanceolate, acute Oblanceolate,

acute

Narrowly

elliptic,

acute

Lip Obscurely

3-lobed in

the middle

Obscurely

3-lobed in

apical half

3-lobed in the

middle

3-lobed in the

middle

3-lobed in the middle 3-lobed in

apical half

3-lobed in

apical half

Lip claw Glabrous Pubescent Glabrous or

sparsely

pubescent

Glabrous or

sparsely

pubescent

Glabrous Densely

pubescent

Densely

pubescent

Lip

middle

lobe

Oblong,

pubescent,

margins

irregularly

toothed

Much larger than

lateral lobes,

broadly ovate

to orbicular,

apiculate

Suborbicular,

margins

erose,

apiculate

Orbicular,

acute

Orbicular-ovate, acute Rotund-

quadrate,

apiculate

Transversely

widely

oblong,

crenulate,

4-lobed,

apiculate

Lip

callus

Small, at

base of

middle

lobe, fleshy

Fleshy transverse

ridge betweenlateral lobes

Fleshy,

between

lateral lobes,

porrect, 2–3-

toothed at

front

Comparatively

small,

centrally

placed

Broad, 3-toothed, just

below insertion of

middle lobe and

extending on the bases

of lateral lobes

Obscure,

fleshy,

between

lateral lobes

Conspicuous,

fleshy

oblong ridge

between

lateral lobes

A phylogenetic and morphological study

123

The lip is squeezed in the middle and furnished with a

prominent fleshy callus.

Discussion

Both plastid and nuclear ITS data indicate common origin

of the species representing the section Superpositae.

According to the results of the DNA analyses the most

closely related species to Polystachya bamendae is P.

lindblomii. The results of morphological analysis support

the relation. Both species are erect or pendulous epiphytic

herbs forming clumps with linear to linear-lanceolate

leaves. The inflorescence of P. bamendae is usually longer

than leaves and that of P. lindblomii much shorter, up to

3 cm long. Flowers of both species are more or less

pubescent with prominent mentum; however, they are

easily distinguishable by the lip structure. The labellum of

Polystachya bamendae is as wide as short, the lip lateral

lobes are prominent up to 2.5 mm long and 2 mm wide, the

middle lobe is transversely widely oblong, 4-lobed, emar-

ginate with a prominent apex on the back side and the

callus is fleshy bilobed ridge along the midnerve in the

middle of the lip. On the contrary the lip of Polystachya

lindblomii is twice longer than wide, the lateral lobes are

small up to 0.6 mm long and 1 mm wide, the middle lobe

is broadly ovate to orbicular and the lip callus is a trans-

verse tridentate fleshy ridge.

The change of frameshift and an occurrence of the stop

codon as the consequence are caused by a presence of indel

19 bp within the sequence of matK gene of the species of

Superpositae (excluding Polystachya lindblomii) and in the

newly described P. bamendae as well. This character is a

molecular synapomorphy and along with morphological

characters they prove a common origin of the representa-

tives of Superpositae. Russell et al. (2010b) suggest that

the matK gene of the mentioned taxa is a pseudogene.

However, considering its function in post-transcriptional

Fig. 5 The distribution map of

Polystachya Hook. section

Superpositae Kraenzl

J. Mytnik-Ejsmont et al.

123

modification process, matK is essential for proper organism

working. Barthet and Hilu (2007) basing on an analysis of

matK cDNA translation did not find the stop codons in

Spathoglottis plicata Blume (Orchidaceae), which were

noted during the DNA analysis of this gene (Freudenstein

et al. 2004). An analogical situation may concern the

supposed pseudogene in Superpositae. Two alternative

hypotheses supporting functionality of the matK gene in

the mentioned species may be presented: (1) RNA editing

mechanisms can improve the reading frame in species with

frameshift indels and premature stop codons; (2) down-

stream frameshift by one base pair eliminate all stop

codons in matK gene in all mentioned species. In this case

ATA codon (for isoleucine) could be initial one. ATA and

ATT (both codons for isoleucine) were reported as start

codon for Thalassiosira pseudonana Cleve and Phaeo-

dactylum tricornutum Bohlin (Chromalveolata), respec-

tively (Imanian et al. 2010).

The question is whether the taxa possessing the

duplication are the monophyletic group? The problem is

interesting, because Polystachya lindblomii, apart from

all other species of the section Superpositae, does not

possess the duplication and possessing the indel seems to

be a new character. If we had more samples of different

representatives of Polystachya londblomii, would the

indel appear? We had an access to a relatively small

amount of samples and therefore we are not able to

unequivocally state it is a constant character, it may be a

result of variability within a population or a molecular

synapomorphy as well. A hypothesis is the duplication of

this fragment took place in the ancestor of all species

possessing today the duplication, what makes the group

monophyletic under condition that Polystachya londblo-

mii is no longer classified within the group. What is

surprising, the new species is the most closely related to

P. lindblomii. The analysis of morphological characters

of P. lindblomii and other species of the section does not

support the hypothesis. To solve the problem and

determine the status of indel (synapomophy or not), an

access to more samples and results of population studies

are needed.

The subsectional division proposed by Mytnik-Ejs-

mont (2011) does not correlate with the results of the

present molecular analyses. Only the nominal subsection

is monophyletic, whereas representatives of the rest two

subsections (Polystachya spatella and P. kermesina) form

strongly supported monophyletic clade. The newly

described Polystachya bamendae forms a weakly sup-

porting clade with P. lindblomii (subsection Spatellae),

however, in the analysis of the ITS marker only. The

plastid marker does not support such relation between

the two species. However, it is noteworthy that only a

few species were analyzed, less than 50 percent of the

representatives of the subsections Spatellae and Super-

positae. Therefore, basing on such weakly representing

test, there is too early in our opinion to verify the

subsectional division proposed by Mytnik-Ejsmont

(2011). Undoubtedly, we may state that Polystachya

lindblomii is an isolated species within the section.

As most species of the section Superpositae present

restricted distribution, they should be subjected to careful

conservation action. These species are very fragile in

their ecosystems and can easily be driven to extinction

(Munishi et al. 2004). At this time we do not have

enough information to discuss the conservation status of

Polystachya bamendae due to the paucity of the popu-

lation. It is known so far from a single locality in

Cameroon and a field survey needs to be carried out to

ascertain the distribution range of the species and pro-

pose the IUCN category.

Acknowledgments We wish to thank the curators and staff of the

cited herbaria for their kind hospitality and assistance during our

visits (B, BM, BR, BRLU, C, HBG, K, L, MA, MO, P, W and WAG).

Fig. 6 The distribution map of Polystachya bamendae Szlachetko,

Baranow & Mytnik

A phylogenetic and morphological study

123

We are very grateful to prof. Tomasz Osiejuk and his research team

(Adam Mickiewicz University, Poland) for their invaluable help

during our field work in Cameroon. We would also like to express our

gratitude to Anton Russell for an access to his DNA sequences and

stimulating discussions. We are also obliged to an anonymous

reviewer for his comments and improving the manuscript. This study

was supported by the grants from the Ministry of Science and Higher

Education (N N303 343735 and 8124/B/PO1/2011/40). This article

was also prepared thanks to the grants from European Commission’s

Research Infrastructure Action via the SYNTHESYS Project at the

Real Jardin Botanico (CSIC; ESTAF 4129), Natural History Museum

of Vienna (AT-TAF 1690, AT-TAF 3903), National Botanic Garden

of Belgium (BE-TAF 750), National Herbarium of The Netherlands

(NL-TAF 2834, NL-TAF 2265), Natural History Museum of Den-

mark (DK-TAF 3231, DK-TAF 4040), and Botanic Garden and

Botanic Museum in Berlin-Dahlem (DE-TAF 3906).

Open Access This article is distributed under the terms of the

Creative Commons Attribution License which permits any use, dis-

tribution, and reproduction in any medium, provided the original

author(s) and the source are credited.

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